Increases in the free, intracellular Ca++ concentration occur under normal conditions in nerve cells and play an important role in the regulation of membrane processes. How Ca++ acts as an intracellular messenger and regulator and how Ca++ is regulated is far from clear. We propose to use a Ca++ ion selective microelectrode and the metallochromic indicator dye arsenazo III to measure changes in the free, intracellular Ca++ concentration in giant molluscan neurons where our preliminary studies show such measurements can be made. There are four specific aims of the proposed research. 1) to determine the intracellular Ca++ concentration using an ion selective microelectrode under basal conditions and after altering Ca++ sequestration or extrusion, or both, 2) To compare transient changes in Ca++ concentration measured by a Ca++ sensitive microelectrode to those measured spectrophotometrically using the dye arsenazo III. 3) To measure the Ca++ concentration at different depths from the inner membrane surface following Ca++ influx through voltage dependent Ca++ channels using a Ca++ sensitive microelectrode and the average change throughout the cell using arsenazo III. These measurements will be compared to the total amount of Ca++ which enters the cell as estimated from the isolated Ca++ current. 4) To investigate the effects of altering the resting level of intracellular Ca++ upon transient changes in Ca++ concentration and upon the isolated Ca++ and Ca++ activated K+ currents. These experiments will provide new and important information about how Ca++ is regulated and may be important for our eventual understanding of disorders of the human central nervous system.